1. Field of the Invention
This invention relates to an improved method for purifying insulin-like growth factor (IGF-I) from variants, impurities, and contaminants associated therewith, particularly when it is produced by bacterial fermentation.
2. Description of Related Art
The production of large quantities of relatively pure, biologically active polypeptides and proteins is important economically for the manufacture of human and animal pharmaceutical formulations, enzymes, and other specialty chemicals. For production of many proteins, recombinant DNA techniques have become the method of choice because large quantities of exogenous proteins can be expressed in bacteria and other host cells. The expression of proteins by recombinant DNA techniques for the production of cells or cell parts that function as biocatalysts is also an important application.
Human insulin-like growth factor-I (IGF-I) is a 7649-dalton polypeptide with a pI of 8.4 [Rinderknecht and Humbel, Proc. Natl. Acad. Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253: 2769 (1978)] belonging to a family of somatomedins with insulin-like and mitogenic biological activities that modulate the action of growth hormone. Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974); Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem., 25: 49 (1986). IGF-I has hypoglycemic effects similar to insulin but also promotes positive nitrogen balance. Underwood et al., Hormone Res., 24: 166 (1986); Guler et al., N. Eng. J. Med., 317: 137 (1987). Due to this range of activities, IGF-I is being tested in humans for uses ranging from wound healing to the reversal of whole body catabolic states. Guler et al., Proc. Natl. Acad. Sci. USA, 85: 4889 (1988).
Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the recombinant protein. The prokaryote E. coli is favored as host because it can be made to produce recombinant proteins in high yields. Numerous U.S. patents on general bacterial expression of DNA encoding proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; and U.S. Pat. No. 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I.
Genetically engineered bio-pharmaceuticals are typically purified from a supernatant containing a variety of diverse host cell contaminants. There is a need in the art for an efficient protocol for selectively separating IGF-I from other molecules, particularly other hydrophobic polypeptides, in the process of purifying IGF-I from a fermentation broth, particularly since the final process pool contains several variant species of recombinant, human IGF-I (rhIGF-I) that are difficult to separate.
IGF-I has been purified using gel filtration followed by ion-exchange chromatography on a sulfopropyl-substituted cation-exchange column, followed by buffer exchange and fractionation by a second gel filtration step. Next, preparative isoelectric focusing further separated the IGF-I from contaminants with similar isoelectric points, followed by two reversed-phase chromatography steps to obtain pure IGF-I. Cornell et al., Prep. Biochem., 14: 123 (1984). Clearly, because of the large number of steps involved, this protocol is relatively inefficient.
An alternative protocol for purifying IGF-I requires the fusion of a Protein A fragment to IGF-I by a linker, where the culture supernatant is passed through an affinity column consisting of IgG coupled to agarose. The IGF-I fusion product binds to the column while contaminants pass through and the bound material is eluted, treated to remove the linker, and passed through IgG-agarose to remove the free Protein A. See Moks et al., Bio/Technology, 5: 379-382 (1987); Sofer, Bio/Technology, 4: 712-715 (1986).
IGF-I has been also purified by a series of adsorption-desorption steps employing a combination of cation-exchange and hydrophobic-interaction adsorbents. See U.S. Pat. No. 5,231,178 issued Jul. 27, 1993. See also KR 9208377 published Sep. 26, 1992 for another method for purifying IGF-I.
Procedures utilizing reversed-phase high performance liquid chromatography (RP-HPLC) have been published for many molecules [Bidlingmeyer, ed., Preparative Liquid Chromatography (Elsevier, Amsterdam, 1987)]. Irreversible binding of insulin and proinsulin to C18 stationary phases has recently been reported [Linde and Welinder, J. Chromatogr., 536: 43 (1991)], with the C4 alkyl chain substitution being preferred to maximize product recovery. Nice et al., J. Chromatogr., 218: 569 (1981). Such RP-HPLC procedures are included in the purification of native IGF-I, synthetic IGF-I, and rhIGF-I. Svoboda et al., Biochemistry, 19: 790 (1980); Petrides et al., Endocrinology, 118: 2034 (1986); Cornell and Brady, J. Chromatogr., 421: 61 (1987); Francis et al., Endocrinology, 124: 1173 (1989). However, preparative RP-HPLC isolation of a recombinant protein resulting in pharmaceutical purity and high yield has eluded the art. Kroeff et al., J. Chromatogr., 461: 45 (1989). Furthermore, the typical pH at which such purification step is operated using a mobile phase is an acid pH, generally around pH 2-3 using trifluoroacetic acid.
Accordingly, it is an object of this invention to provide an improved process for separating IGF-I from its related fermentation variants by means of reversed-phase liquid chromatography.
It is another object to provide a process for purifying IGF-I from related variants using elution conditions involving physiological pH.
It is still another object to provide a process for purifying IGF-I that results in considerable improvement in its homogeneity.
These and other objects will be apparent to those of ordinary skill in the art.